Ab105731

The treated cells were washed twice with cold 1X phosphate-buffered saline, lysed with RIPA lysate buffer for 20 min, and centrifuged (12,000 rpm, 10 min, 4°C) to obtain the supernatant. The loading buffer was added, and the supernatant was denatured at 95°C for 5 min until protein denaturation was achieved. Electrophoresis analysis of protein samples (10 μL/well) was performed using 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Proteins were transferred onto nitrocellulose membranes (Millipore, Burlington, MA, USA). The blots were blocked with 5% nonfat milk powder for 1 h and incubated with corresponding primary antibody at 4°C overnight. On the second day, the blots were cleaned with 1X tris-buffered saline and incubated with the appropriate secondary antibodies at room temperature for 1 h. Protein brands were detected by chemiluminescence (Millipore, Burlington, USA), and the expression level was determined using Image J.
Primary antibodies against JAK (ab133666), anti-p-JAK (ab138005), anti-STAT1 (ab109320), anti-p-STAT1 (ab109461), mTOR (ab32028), p-mTOR (ab109268), p-AKT (ab105731), Bax (ab32503), and Bcl2 (ab182858) were purchased from Abcam (Cambridge, UK). The following secondary antibodies were purchased from GenScript (Piscataway, NJ, USA): goat anti-rabbit IgG (H&L) and goat anti-mouse IgG (H&L).

Total proteins were extracted using RIPA buffer (50-mM Tris (pH 7.4), 1-mM EDTA, 150-mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemental with protease inhibitors (Roche). Antibodies against STEAP4 (ab113230, Abcam), p-AKT (ab105731, Abcam), AKT (#9272, CST), and GAPDH (G8795, Sigma) were used. GAPDH was used as the loading control.

Cytospin slides (2 x 10⁴ cells/slide) were prepared using NewSilane II Micro Slides (Muto Pure Chemicals, Tokyo, Japan), fixed with 4% PFA at room temperature for 10 min, blocked with Blocking One Histo (Nacalai Tesque, Kyoto, Japan) at room temperature for 2 h, and then stained with primary antibodies to human PD-L1 (1:200 dilution, #ab205921, Abcam, Cambridge, MA, USA), PD-L2 (1:500, #MAB1224, R&D Systems, Minneapolis, MN, USA), Akt (1:200, #9272, Cell Signaling, Danvers, MA, USA) and phospho-Akt (1:200, #ab105731, Abcam) at 4°C for overnight, coupled with secondary antibodies to Alexa Fluor® 594 goat anti-rabbit IgG (1:300, #A11072, Thermo Fisher Scientific) and anti-mouse IgG (1:300, #A11005, Thermo Fisher Scientific) at room temperature for 3h. For nucleus staining, we used DAPI (0.4 μg/ml, #D9564, SIGMA). The immunofluorescence was examined with a confocal microscope (LSM-800, Zeiss, Oberkochen, Germany) and staining intensity was quantitated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).